Part:BBa_K5127009

Brer-pBrer-gfp

This composite part combines the BreR transcriptional regulator and the GFP after the pBreR to enable the characterization of the system.

Team: BNDS-China 2024

Our team aims to detect the intake of food before activating our comprehensive platform that regulates levels of gut metabolites, with DCA (a subtype of bile acid) as the indicator of food presence. Within this framework, the BreR system stands out as an effective food sensor for detecting DCA, and the response range best aligns with the fluctuations of DCA levels in human intestines.

Structural analysis of BreR repressor

As expected, BreR structure predicted by alphafold 3 (Abramson et al., 2024) suggests a homodimer oligomer (Figure 1A). Moreover, the protein-DNA interaction prediction by alphafold 3 accurately predicted the BreR binding to the BreO operator surrounded by the upstream spacer and downstream promoter DNA (Figure 1B). Structural alignment by PyMOL cealign function showed a significant structural shift upon DNA binding (Figure 1C, red circled). Taken together, the structural evidence from alphafold 3 further the DNA binding capability of BreR, and we thus adopted this allosteric TetR-family transcription factor for DCA biosensor design.

Figure 1. The structure predicted by alphafold 3. (A) The apo-structure of BreR homodimer (chain A: cyan; chain B: green). (B) The DNA-bound structure of BreR-BreO flanked by upstream spacer and downstream promoter DNA. (C) Structural alignment of BreR apo-structure and DNA-bound structure; a structural shift was observed at the helix-turn-helix DNA binding domain (red circled).

Characterization of DCA biosensor using BreR

In our design, we used constitutive promoter J23106 for BreR expression. The pBreR is designed with an optimized version of the J23101 promoter with BreO operator incorporated (Beabout et al., 2023) (Figure 2). We inserted GFP downstream pBreR as the reporter gene.

Figure 2. Plasmid design of BreR-pDCA. Created by biorender.com.

We used Golden Gate Assembly to construct pBreR plasmid. PCR and Gel Electrophoresis were performed to verify the materials of the assembly (Figure 3).

Figure 3. The AGE result of the PCR products of materials to construct pBreR plasmid.

To evaluate the inducibility of the downstream gene, two cultures of E. coli transformed with the BreR plasmid were prepared, with DCA added to one. The DCA-treated culture exhibited strong green fluorescence, qualitatively demonstrating that our design is responsive to DCA (Figure 4).

Figure 4. Observation of pBreR with DCA+ (left) or DCA- (right). The left bacteria culture with DCA treatment showed strong green fluorescence.

We quantified the relationship between DCA concentration and pBreR promoter activity. The dynamic range of BreR biosensor system was about 6-fold with the maximum signal achieved at 500μM DCA; above 500μM, the change in promoter activity became subtle (Figure 5).

Figure 5. Quantitative relationship between DCA concentration and fluorescence level. The GFP expression was quantified by measuring fluorescence / ABS600.

Further, we characterized the kinetic behavior of this biosensor. A gradient of DCA concentration was added into bacterial cultures, and fluorescence / ABS600 values were measured over time using a plate-reader to assess promoter activity (Figure 6).

Figure 6. A kinetic assay for GFP expression was conducted over 18 hours using various DCA concentrations. GFP expression was quantified by measuring fluorescence / ABS600.

The results aligned with our expectations, in which bacteria cultures added with higher DCA concentration had stronger GFP expression levels. The dynamic range of BreR sensor system was about 5-fold at 1000μM DCA, much higher compared to that of pDCA_VFA0359 system (about 2-fold).

This trend was especially clear when DCA concentration was above 62.5μM. When DCA concentration was below 62.5μM, the difference in DCA concentration between groups might be too small to be reflected in significant difference in GFP expression.

Applying DCA biosensor for butyrate production

To implement our overall design, we placed Tes4 downstream to promoter pBreR to make the butyrate production responsive to the food availability. In this way, the presence of bile acid will trigger the expression of Tes4 and produce butyrate. We replaced the GFP reporter gene in the BreR-mediated DCA biosensor with the coding frame of Tes4 (Figure 7).

Figure 7. Plasmid design of BreR-Tes4. Created by biorender.com.

We used Golden Gate Assembly to construct BreR-Tes4 from BreR backbone and Tes4 fragment. PCR and Gel Electrophoresis were performed to verify the success in constructing each component and the full plasmid (Figure 8).

Figure 8. The AGE results of the PCR products of BreR construction. A and B, materials to construct BreR-Tes4. C, Golden gate assembly result of BreR-Tes4 construction. The band at 4372bp in (C) indicated the success in plasmid construction.

We performed SDS-PAGE to verify the expression of Tes4. The band representing Tes4 (19.5kDa) was thicker in the DCA-treated group, showing the successful induction of Tes4 by DCA (Figure 9).

Figure 9. The SDS-PAGE result of successful Tes4 expression induced by DCA.

We are trying to further validate the production of butyrate with GC-MS assay to find out if there is any butyrate produced.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 400
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 653
  Illegal NheI site found at 676
  Illegal PstI site found at 400
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 400
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 400
  Illegal AgeI site found at 853
  
• 1000
  COMPATIBLE WITH RFC[1000]